Project Summary Staphylococcus aureus is a major human pathogen that causes epicutaneous and subcutaneous skin infections. Evidence shows that S. aureus could be a key contributor to chronic itch conditions, including atopic dermatitis (AD), where over 90% of sufferers are colonized by S. aureus. We hypothesize S. aureus directly activates pruriceptor neurons, the itch-mediating sensory neurons densely innervating the epidermis, during epicutaneous skin infections. In turn, this exacerbates and promotes not only itch, but also skin inflammation and disease pathology in AD. Sensory neuron activation and the subsequent release of neuropeptides cause neurogenic inflammation, resulting in the recruitment/activation of immune cells. The main objective of this proposal is to determine the mechanisms of itch during S. aureus epicutaneous infection and the contribution of sensory neurons in driving inflammation and skin pathology. In Specific Aim 1, we will establish a mouse model of epicutaneous S. aureus infection where chronic itch behavior can be quantified. Nerve sprouting and nerve density into the epidermis, key components of AD, will also be analyzed. I have recently shown S. aureus pore-forming toxins (PFTs), such as alpha hemolysin (Hla), are capable of activating sensory neurons. Several recent studies using this model of infection have pointed to the key role of PSM?s, a class of S. aureus PFTs, in inflammation. We have obtained isogenic mutant strains of bacteria lacking these toxins and will test if the specific PFTs, Hla and PSM?s, contribute to itch-behavior in vivo. In Specific Aim 2, we will determine whether these sensory neurons mediate inflammation and host-defense during an epicutaneous S. aureus infection. Sensory neurons will be depleted using chemical and transgenic strategies. Trpv1 expressing neurons will be depleted using resiniferatoxin and by using the transgenic mouse line, Trpv1-Cre/Dta. In addition, to target a different, but overlapping set of sensory neurons, the transgenic mouse line Nav1.8- cre/Dta will be used. In these strategies, the subsequent consequences on immune cell recruitment, bacterial clearance, and skin pathology will be determined. The proposed research is novel given the poor mechanistic understanding of pathogen-induced itch, particularly in S. aureus infection. The results will not only fill a critical gap in our knowledge of direct interactions of S. aureus with sensory neurons to drive itch, but also pioneer studies determining the role of the nervous system in driving inflammation in the skin. Therapeutic strategies targeting these newfound bacterial-neuron interactions can be developed as novel treatments to alleviate both AD pathology and its debilitating itch, especially in conjunction with S. aureus infection.